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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌 | |
| dc.contributor.author | Jyun-Yu Yao | en |
| dc.contributor.author | 姚俊宇 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:23:11Z | - |
| dc.date.available | 2019-07-07 | |
| dc.date.copyright | 2017-07-07 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-06-23 | |
| dc.identifier.citation | REFERENCES
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Bajda, A., Konopka-Postupolska, D., Krzymowska, M., Hennig, J., Skorupinska-Tudek, K., Surmacz, L., Wojcik, J., Matysiak, Z., Chojnacki, T., Skorzynska-Polit, E., Drazkiewicz, M., Patrzylas, P., Tomaszewska, M., Kania, M., Swist, M., Danikiewicz, W., Piotrowska, W., and Swiezewska, E. (2009) Role of polyisoprenoids in tobacco resistance against biotic stresses. Physiol Plant 135, 351-364 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59419 | - |
| dc.description.abstract | 異戊二烯轉移酶 (prenyltransferase)中有一種類的功能是催化將特定數量的異戊烯焦磷酸 (isopentenyl diphosphate, IPP)與法尼基焦磷酸 (farnesyl diphosphate, FPP)進行連續縮合反應，將碳鏈延長到指定的長度。根據受質異戊烯焦磷酸酵素反應時所形成的雙鍵立體化學，而將異戊二烯轉移酶分類為順式和反式。順式異戊二烯轉移酶主要合成較長的產物作為脂質載體，主導真核生物中糖蛋白或細菌中肽聚醣的生物合成。然而因為植物中的順式異戊烯基轉移酶通常存在幾種同源亞型，所以功能較少被仔細了解。來自麝香百合花的順式異戊烯基轉移酶基因LLA66是在絨氈層和小孢子中，所鑑定的第一個異戊烯基轉移酶。本次研究中，我們利用在釀酒酵母 (Saccharomyces cerevisiae)表現重組蛋白LLA66，發現它是一種獨特的中鏈萜類合成酶。藉由高效液相色譜法 (HPLC)及薄層色譜 （TLC）分析中，顯示產物為45個碳鏈的萜類。此外在使用Ni-NTA親和層析純化有6個His標記的LLA66蛋白時，我們還發現純化後的蛋白質溶液中具有將IPP轉化為法尼醇(farnesol)的催化活性。通過進一步的數據，我們認為是IPP：DMAPP異構酶、FPP合成酶和鹼性磷酸酶。但它們是否會形成蛋白質複合物是需要實驗的進一步驗證。 | zh_TW |
| dc.description.abstract | A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths by consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). According to the stereochemistry of the double bonds formed by IPP condensation, these prenyltransferase are classified as cis- and trans-types. Cis-prenyltransferases synthesize longer products as lipid carriers to mediate biosynthesis of peptidoglycan in bacteria or glycoproteins in eukaryotes. However, the functions of cis-prenyltransferases in plants are less understood because there are several homologous isoforms in different compartments. The cis-prenyltransferase gene LLA66 from Lilium longiflorum anther is the first prenyltransferase identified in the tapetum and microspores. As reported here, we have produced the recombinant LLA66 in Saccharomyces cerevisiae, performed in-vitro characterization of the enzyme and found it is a unique medium-chain synthase, and was resolved C45 isoprenoids in thin layer chromatography (TLC) and HPLC. After purification of the hexa-His-tagged LLA66 using Ni-NTA affinity chromatography, we also identified enzyme activities to convert IPP into farnesol in the purified protein mixture. By further analysis, we thought that a protein mixture of IPP:DMAPP isomerase, FPP synthase and alkaline phosphatase was co-purified with LLA66. We will further characterize the protein complex. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:23:11Z (GMT). No. of bitstreams: 1 ntu-106-R04b46008-1.pdf: 2909242 bytes, checksum: cc4b0eee4a1f30722b2d082e3dc76f75 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | LITS OF TABLE
中文摘要 iv ABSTRACT v LITS OF TABLE vi LIST OF FIGURE vii ABBREVIATIONS ix 1. INTRODUCTION 1 1.1 Isoprenoid 1 1.2 Isoprenyl pyrophosphate synthases 1 1.3 In this study 2 MATERIALS AND METHODS 4 2.1 Chemicals 4 2.2 Preparation of LLA66 construct. 4 2.2.1 pYES2/CT-LLA66 construct for S.cerevisiae 5 2.2.2 LLA66 construct for E. coli 5 2.3 Extraction of polyisoprenoids from yeast 5 2.4 HPCL/UV analysis of polyisoprenoids. 6 2.5 Purification of His-tagged LLA66 7 2.5.1 Overexpression of LLA66 in S. cerevisiae 7 2.5.2 Overxpression of LLA66 in E. coli 8 2.6 Activity assay of LLA66 9 2.6.1 EnzChek Pyrophosphate assay 9 2.6.2 Radiative [14C] IPP assay 9 2.7 TLC analysis of polyisoprenoids 11 2.8 Ion exchange chromatography using Mono-S and Mono-Q columns 11 RESULTS 13 3.1 Functional characterization of LLA66 activity in S. cerevisiae rer2Δ mutant 13 3.2 HPLC/UV analysis of polyisoprenoids of Saccharomyces cerevisiae 13 3.3 LLA66 overexpression and purification in Saccharomyces cerevisiae 14 3.4 Unexpected activity of LLA66 from Saccharomyces cerevisiae expression 14 3.5 Thin layer chromatography analysis of polyisoprenoids of LLA66 15 3.6 Identification of Saccharomyces cerevisiae enzyme mixture for unexpected IPP-utilizing activity 15 3.7 Ion exchange chromatography analysis of LLA66 and yeast enzyme 17 3.8 LLA66 overexpression and purification in E. coli 17 3.9 LLA66 activity assay from E. coli expression 18 3.10 Characterization and kinetics of LLA66 enzyme 19 DISCUSSION 20 TABLES 24 FIGURES 30 REFERENCES 58 | |
| dc.language.iso | en | |
| dc.title | 釀酒酵母中表現並鑑定植物順式異戊二烯轉移酶LLA66 | zh_TW |
| dc.title | Expression and characterization of a plant cis-prenyltransferase LLA66 in Saccharomyces cerevisiae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊啟伸,郭致榮 | |
| dc.subject.keyword | 異戊二烯轉移?,麝香百合,釀酒酵母,中鏈?類, | zh_TW |
| dc.subject.keyword | prenyltransferase,Lilium longiflorum,Saccharomyces cerevisiae,medium-chain isoprenoid, | en |
| dc.relation.page | 65 | |
| dc.identifier.doi | 10.6342/NTU201701044 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-06-23 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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